Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

Mammalian-wide interspersed repeats (MIRs) are retrotransposed elements of mammalian genomes. Here, we report the specific binding of zinc finger protein ZNF768 to the sequence motif GCTGTGTG (N20) CCTCTCTG in the core region of MIRs. ZNF768 binding is preferentially associated with euchromatin and promoter regions of genes. Binding was observed for genes expressed in a cell type-specific manner in human B cell line Raji and osteosarcoma U2OS cells. Mass spectrometric analysis revealed binding of ZNF768 to Elongator components Elp1, Elp2 and Elp3 and other nuclear factors. The N-terminus of ZNF768 contains a heptad repeat array structurally related to the C-terminal domain (CTD) of RNA polymerase II. This array evolved in placental animals but not marsupials and monotreme species, displays species-specific length variations, and possibly fulfills CTD related functions in gene regulation. We propose that the evolution of MIRs and ZNF768 has extended the repertoire of gene regulatory mechanisms in mammals and that ZNF768 binding is associated with cell type-specific gene expression.

MYC is an unstable protein, and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that MYC proteasomal turnover is dispensable for loading of RNA polymerase II (RNAPII). In contrast, MYC turnover is essential for recruitment of TRRAP, histone acetylation, and binding of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII and transcriptional elongation. In the absence of histone acetylation and P-TEFb recruitment, MYC associates with the PAF1 complex (PAF1C) through a conserved domain in the MYC amino terminus ("MYC box I"). Depletion of the PAF1C subunit CDC73 enhances expression of MYC target genes, suggesting that the MYC/PAF1C complex can inhibit transcription. Because several ubiquitin ligases bind to MYC via the same domain ("MYC box II") that interacts with TRRAP, we propose that degradation of MYC limits the accumulation of MYC/PAF1C complexes during transcriptional activation.

Cyclin-dependent kinase 12 (CDK12) phosphorylates RNA Pol II C-terminal domain (CTD) to promote transcriptional elongation of large DNA damage response genes. CDK12 is frequently mutated or amplified in cancer and its loss sensitises cells to DNA damage. Here we present 3 crystal structures of the human CDK12/CycK complex including apo, AMP-PNP and covalent inhibitor complexes. Kinase assays compare domain truncations and report the Km values for substrate. THZ531 is presented as a potent and selective inhibitor of CDK12 with nanomolar activity in leukemic cell lines.

Although poly (ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after restoration of HR. Here, we report that dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 5, and 9, additionally has potent activity against CDK12, a transcriptional regulator of HR. In BRCA-mutated triple-negative breast cancer (TNBC) cells and patient-derived xenografts (PDXs), dinaciclib ablates restored HR and reverses PARP inhibitor resistance. Additionally, we show that de novo resistance to PARP inhibition in BRCA1-mutated cell lines and a PDX derived from a PARP-inhibitor-naive BRCA1 carrier is mediated by residual HR and is reversed by CDK12 inhibition. Finally, dinaciclib augments the degree of response in a PARP-inhibitor-sensitive model, converting tumor growth inhibition to durable regression. These results highlight the significance of HR disruption as a therapeutic strategy and support the broad use of combined CDK12 and PARP inhibition in TNBC.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5'-to-3' "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

The bromodomain protein Brd4 regulates the transcription of signal-inducible genes. This is achieved by recruiting the positive transcription elongation factor P-TEFb to promoters by its P-TEFb interaction domain (PID). Here we show that Brd4 stimulates the kinase activity of P-TEFb for phosphorylation of the C-terminal domain (CTD) of RNA polymerase II over basal levels. The CTD phosphorylation saturation levels, the preferences for pre-phosphorylated substrates, and the phosphorylation specificity for Ser5 of the CTD however remain unchanged. Inhibition of P-TEFb by Hexim1 is relieved by Brd4, although no mutual displacement with the Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence motif similarity to the trans-activating Tat protein from HIV-1, which includes a core RxL motif, a polybasic cluster known as arginine-rich motif, and a C-terminal leucine motif. Mutation of these motifs to alanine significantly diminished the stimulatory effect of Brd4 and fully abrogated its activation potential in presence of Hexim1. Yet the protein was not found to bind Cyclin T1 as Tat, but only P-TEFb with a dissociation constant of 0.5 μM. Our data suggest a model where Brd4 acts on the kinase subunit of P-TEFb to relieve inhibition and stimulate substrate recognition.